Blakley R L, Appleman J R, Freisheim J H, Jablonsky M J
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
Arch Biochem Biophys. 1993 Nov 1;306(2):501-9. doi: 10.1006/abbi.1993.1543.
Nuclear magnetic resonance (NMR) spectra for [2-amino,3-15N2]folate and [2-13C]folate complexed with human dihydrofolate reductase, and for complexes of similarly labeled dihydrofolate, show that the N-3 proton of bound folate or dihydrofolate exchanges slowly with solvent and that the bound substrates are in the imino-keto tautomeric form. Previously proposed schemes for substrate protonation that require bound substrate to be in the enolic tautomer are therefore unlikely. The NMR spectra for bound folate are unchanged by raising the pH from 7 to 9.5, whereas those for free folate show marked changes due to ionization for the N-3 proton. The fraction of bound folate with the N-3 proton ionized at pH 9.5 is therefore very small, and the rate constant for the dissociation of the ionized species must be at least 320 times faster than for the protonated species. Comparison of NMR spectra over the pH range 5 to 7 gives no indication of a change in ionization state of the Glu30 carboxyl group over this pH range. This raises doubts about whether the apparent pKa of approximately 6 that describes pH dependence of hydride transfer is due to ionization of this carboxyl group.
与人类二氢叶酸还原酶复合的[2-氨基，3-¹⁵N₂]叶酸和[2-¹³C]叶酸以及类似标记的二氢叶酸复合物的核磁共振（NMR）光谱表明，结合态叶酸或二氢叶酸的N-3质子与溶剂的交换缓慢，并且结合态底物处于亚氨基-酮互变异构形式。因此，先前提出的要求结合态底物处于烯醇互变异构体的底物质子化方案不太可能成立。将pH从7提高到9.5时，结合态叶酸的NMR光谱没有变化，而游离叶酸的NMR光谱由于N-3质子的电离而显示出明显变化。因此，在pH 9.5时N-3质子电离的结合态叶酸的比例非常小，并且电离物种的解离速率常数必须至少比质子化物种快320倍。在pH范围5至7内对NMR光谱的比较没有表明在此pH范围内Glu30羧基的电离状态发生变化。这引发了人们对于描述氢化物转移的pH依赖性的表观pKa约为6是否归因于该羧基电离的怀疑。
